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(EST)
Printed by: Nguyen Nhan Official Date: Official as of 01-May-2020
Azithromycin for Injection
DEFINITION
Azithromycin for Injection is a sterile, dry mixture of
azithromycin and a suitable stabilizing agent. It contains
NLT 90.0% and NMT 110.0% of the labeled amount of
azithromycin (C38H72N2O12).
IDENTIFICATION
• A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
Change to read:
▲
• B. ▲SPECTROSCOPIC IDENTIFICATION TESTS á197ñ, Infrared
Spectroscopy: 197A▲ (CN 1-May-2020)
Standard: 25 mg/mL of USP Azithromycin RS in acetonitrile.
Pass the solution through a suitable filter, and remove the
solvent by natural evaporation.
Sample: Equivalent to 25 mg/mL of azithromycin from
Azithromycin for Injection in acetonitrile. Pass the solution
through a suitable filter, and remove the solvent by natural
evaporation.
Analysis: Examine the spectra of the Standard and the
Sample in the range between 3800 and 650 cm–1.
Acceptance criteria: The Sample exhibits bands at about
900, 995, 1165, 1376, 1456, 1725, and 2936 cm–1 similar
to the spectrum from the Standard similarly
obtained.▲ (USP 1-Aug-2019)
ASSAY
ci
• PROCEDURE
ffi
Buffer: 6.7 mg/mL of dibasic potassium phosphate in water
Mobile phase: Acetonitrile and Buffer (52:48). Adjust with
10 N potassium hydroxide to a pH of 11.0 ± 0.1.
Diluent: Acetonitrile and water (52:48)
System suitability solution: 1 mg/mL each of USP
Azaerythromycin A RS and USP Azithromycin RS in a
O
mixture of acetonitrile and water (52:48). Dissolve first in
acetonitrile, and then dilute with water to volume.
Standard solution: 1 mg/mL of USP Azithromycin RS in a
mixture of acetonitrile and water (52:48). Dissolve first in
acetonitrile, and dilute with water to volume.
Sample solution: Nominally equivalent to 1 mg/mL of
azithromycin from Azithromycin for Injection in Diluent
prepared as follows. Reconstitute 3 vials individually as
directed in the labeling. Mix the contents of all the
reconstituted vials. Dilute a portion of the mixture with
Diluent.
Chromatographic system
(See Chromatography á621ñ, System Suitability.)
Mode: LC
Detector: UV 215 nm
Columns
Guard: 4.6-mm × 1-cm; 5-µm packing L67
Analytical: 4.6-mm × 15-cm; 5-µm packing L67
Temperatures
Autosampler: 15°
Column: 40°
Flow rate: 1 mL/min
Injection volume: 15 µL
System suitability
Samples: System suitability solution and Standard solution
[NOTE—The relative retention times for
azaerythromycin A and azithromycin are 0.68 and
1.0, respectively.]
Suitability requirements
Resolution: NLT 2.5 between azaerythromycin A and
azithromycin, System suitability solution
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Tailing factor: NLT 0.9 and NMT 1.5, Standard solution
Relative standard deviation: NMT 2%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
azithromycin (C38H72N2O12) in the portion of
Azithromycin for Injection taken:
Result = (rU/rS) × (CS/CU) × P × F × 100
rU = peak response from the Sample solution
rS = peak response from the Standard solution
CS = concentration of USP Azithromycin RS in the
Standard solution (mg/mL)
CU = nominal concentration of azithromycin in the
Sample solution (mg/mL)
P = potency of USP Azithromycin RS (µg/mg)
F = conversion factor, 0.001 mg/µg
Acceptance criteria: 90.0%–110.0%
PERFORMANCE TESTS
al
• UNIFORMITY OF DOSAGE UNITS á905ñ: Meets the
requirements
IMPURITIES
[NOTE—The test for Limit of Azithromycin N-Oxide,
Desosaminylazithromycin, and N-Demethylazithromycin
does not quantify aminoazithromycin, formamido
analog, methylformamido analog, and
3′-de(dimethylamino)-3′-oxoazithromycin. If these
impurities are part of the impurity profile, the Limit of
Aminoazithromycin, Formamido Analog,
Methylformamido Analog, and 3′-De(dimethylamino)-
3′-oxoazithromycin test is recommended in addition to
the test for Limit of Azithromycin N-Oxide,
Desosaminylazithromycin, and N-Demethylazithromycin.]
Change to read:
• LIMIT OF AZITHROMYCIN N-OXIDE,
DESOSAMINYLAZITHROMYCIN, AND
N-DEMETHYLAZITHROMYCIN
Buffer: 3.5 g/L of dibasic potassium phosphate
Mobile phase: Acetonitrile and Buffer (23:77). Adjust with
5 N potassium hydroxide to a pH of 10.55 ± 0.05.
Standard stock solution: 0.05 mg/mL of USP Azithromycin
N-oxide RS, 45 µg/mL of USP
Desosaminylazithromycin RS, and 160 µg/mL each of USP
N-Demethylazithromycin RS and USP Azithromycin RS in
acetonitrile. Sonicate if necessary to dissolve.
Standard solution: 0.001 mg/mL of azithromycin N-oxide,
0.9 µg/mL of desosaminylazithromycin, and 3.2 µg/mL
each of N-demethylazithromycin and azithromycin from
Standard stock solution in Mobile phase
Sample solution: Nominally equivalent to 0.3 mg/mL of
azithromycin in Mobile phase from Azithromycin for
Injection
Chromatographic system
(See Chromatography á621ñ, System Suitability.)
Mode: LC
Detector: Amperometric electrochemical
Electrodes: Dual series glassy carbon
Mode: Oxidative screen
Electrode 1: +0.70 ± 0.05 V
Electrode 2: +0.82 ± 0.05 V
▲
▲ (USP 1-Aug-2019)
Column: 4.6-mm × 15-cm; 3-µm packing L49
Autosampler temperature: 5°
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Flow rate: 1 mL/min Diluent: Acetonitrile and water (46:54)

Injection volume: 50 µL Standard stock solution: 0.09 mg/mL of USP
System suitability Desosaminylazithromycin RS, 0.21 mg/mL of USP
Sample: Standard solution N-Demethylazithromycin RS, and 0.30 mg/mL of USP
[NOTE—See Table 1 for relative retention times.] Azithromycin RS in acetonitrile
Suitability requirements Standard solution: 0.0018 mg/mL of
Tailing factor: NMT 2.0 for azithromycin and NMT desosaminylazithromycin, 0.0042 mg/mL of
2.6 for N-demethylazithromycin N-demethylazithromycin, and 0.006 mg/mL of
Relative standard deviation: NMT 10.0% for azithromycin in Diluent
azithromycin N-oxide, desosaminylazithromycin, Sample solution: Nominally equivalent to 0.6 mg/mL of
N-demethylazithromycin, and azithromycin azithromycin from Azithromycin for Injection in Diluent.
Analysis Reconstitute 3 vials individually, as directed in the labeling.
Samples: Standard solution and Sample solution Mix the contents of all the reconstituted vials. Dilute a
Calculate the percentage of each specified impurity in the portion of the mixture with Diluent. The Sample solution
portion of Azithromycin for Injection taken: must be injected immediately after preparation.
Blank: Use the Diluent.
Result = (rU/rS) × (CS/CU) × P × 100 Chromatographic system
(See Chromatography á621ñ, System Suitability.)
rU = peak response of each specified impurity from the Mode: LC
Sample solution Detector: Amperometric electrochemical
rS = peak response of each specified impurity from the Electrodes: Dual series glassy carbon
Standard solution Mode: Oxidative screen
CS = concentration of the relevant impurity Reference

al
Electrode 1: +0.70 ± 0.05 V
Standard in the Standard solution (mg/mL) Electrode 2: +0.82 ± 0.05 V
CU = nominal concentration of azithromycin in the ▲
▲ (USP 1-Aug-2019)
Sample solution (mg/mL) Columns
P = potency of the relevant Reference Standard (mg/ Guard: 4.6-mm × 1-cm; 5-µm packing L67
mg) Analytical: 4.6-mm × 25-cm; 5-µm packing L67
Acceptance criteria: See Table 1. The reporting
▲
threshold▲ (USP 1-Aug-2019) is 0.05%.
ci Temperatures
Autosampler: 15°
Column: 40°
Flow rate: 1 mL/min
Table 1 Injection volume: 25 µL
ffi
Relative Acceptance System suitability
Retention Criteria, Sample: Standard solution
Name Time NMT (%)
[NOTE—See Table 2 for relative retention times.]
Azithromycin N-oxidea 0.17 1.0 Suitability requirements
b
Resolution: NLT 1.5 between desosaminylazithromycin
Desosaminylazithromycin 0.27 0.3
and N-demethylazithromycin
O

Erythromycin A oximec, d 0.35 — Tailing factor: NMT 1.5 for azithromycin

e
Relative standard deviation: NMT 5% for azithromycin
N-Demethylazithromycin 0.50 1.0
Analysis
Azaerythromycin A c, f
0.85 — Samples: Standard solution, Sample solution, and Blank
Disregard any peaks corresponding to those obtained
Azithromycin 1.00 —
from the Blank.
a (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O- Calculate the percentage of each impurity in the portion of
methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy- Azithromycin for Injection taken:
3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylazinoyl)-β-D-
xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. Result = (rU/rS) × (CS/CU) × P × F × 100
b (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-Ethyl-3,4,10,13-tetrahydroxy-
3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-dimethylamino-β-D-xylo-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.
rU = peak response of each impurity from the Sample
c Process impurities that are controlled in the drug substance are not to be solution
reported. They are listed here for information only. rS = peak response of azithromycin from the Standard
d (3R,4S,5S,6R,7R,9R,11S,12R,13S,14R,E)-6-[[3,4,6-Trideoxy-3-(dimeth- solution
ylamino)-β-D-xylo-hexopyranosyl]oxy]-14-ethyl-7,12,13-trihydroxy-4-[(2,6- CS = concentration of USP Azithromycin RS in the
dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-10- Standard solution (mg/mL)
(hydroxyimino)-3,5,7,9,11,13-hexamethyloxacyclotetradecan-2-one.
e (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O- CU = nominal concentration of azithromycin in the
methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy- Sample solution (mg/mL)
3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-methylamino-β-D-xylo- P = potency of USP Azithromycin RS (µg/mg)
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. F = conversion factor, 0.001 mg/µg
f 9-Deoxo-9a-aza-9a-homoerythromycin A.

Acceptance criteria: See Table 2.

Change to read:
Table 2
• LIMIT OF AMINOAZITHROMYCIN, FORMAMIDO ANALOG, Relative Acceptance
METHYLFORMAMIDO ANALOG, AND Retention Criteria,
3′-DE(DIMETHYLAMINO)-3′-OXOAZITHROMYCIN (if present) Name Time NMT (%)
Buffer: 3.5 g/L of dibasic potassium phosphate in water
Erythromycin A iminoethera, b 0.20 —
Mobile phase: Acetonitrile and Buffer (46:54). Adjust with
10 N potassium hydroxide to a pH of 11.0 ± 0.1.

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Table 2 (continued) heptamethyl-11-[[3,4,6-trideoxy-3-oxo-β-D-xylo-hexopyranosyl]oxy]-1-oxa-6-

azacyclopentadecan-15-one.
Relative Acceptance m (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O-
Retention Criteria, methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-
Name Time NMT (%) 3,5,6,8,10,12,14-heptamethyl-11-[[3-[N-(4-acetamidophenylsulfonyl)-N-
3′-(N,N-Didemethyl)azith methylamino]-3,4,6-trideoxy-β-D-xylo-hexopyranosyl]oxy]-1-oxa-6-
- azacyclopentadecan-15-one.
n (2R,3R,4S,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O-
romycin (aminoazithromy-
cin)c + 3′-(N,N-didemethyl)- methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-4,10-dihydroxy-3,5,6,8,10,12,14-
3′-N-formylazithromycin heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]
(formamido analog)d 0.25 1.0 oxy]-1-oxa-6-azacyclopentadecan-15-one.
o Total impurities include desosaminylazithromycin and N-
Azithromycin F a, e
0.30 — demethylazithromycin.

Desosaminylazithromycinf, g 0.31 —
SPECIFIC TESTS
3′-N-Demethyl-3′-N-formyla-
zithromycin Change to read:
(methylformamido analog)h 0.32 1.0
• BACTERIAL ENDOTOXINS TEST á85ñ: ▲
Meets the
N-Demethylazithromycinf, i 0.35 —
requirements▲ (USP 1-Aug-2019)
Erythromycin A oximea, j 0.42 —

—
Change to read:
Azaerythromycin Aa, k 0.63
3′-De(dimethylamino)-3′- • STERILITY TESTS á71ñ: ▲Meets the
oxoazithromycinl 0.72 1.0 requirements▲ (USP 1-Aug-2019)

al
3′-N-Demethyl-
• PARTICULATE MATTER IN INJECTIONS á788ñ: Meets the
3′-N-[(4-methylphenyl)sul- requirements
fonyl] — • PH á791ñ: 6.4–6.8, determined in a solution constituted as
- directed in the labeling
azithromycina, m 0.85
• WATER DETERMINATION á921ñ, Method I: NMT 2.0%
• OTHER REQUIREMENTS: It meets the requirements under
Azithromycin

Azithromycin B
(3-deoxyazithromycin)a, n
1.00

1.64
—

—
ci Injections and Implanted Drug Products á1ñ.
ADDITIONAL REQUIREMENTS
Any other unspecified • PACKAGING AND STORAGE: Preserve as described under
— Packaging and Storage Requirements á659ñ, Injection
impurity 0.2
ffi
Packaging, Packaging for Constitution. Store at controlled
Total impuritieso — 3.0
room temperature.
a Process impurities that are controlled in the drug substance are not to be • LABELING: It meets the requirements for Labeling á7ñ, Labels
reported. They are listed here for information only. and Labeling for Injectable Products.
b (3R,4R,5S,6R,9R,10S,11S,12R,13S,15R,Z)-12-[[3,4,6-Trideoxy-3- • USP REFERENCE STANDARDS á11ñ
(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-6-ethyl-4,5-dihydroxy-10-[(2,6- USP Azaerythromycin A RS
O

dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-3,5,9,11,13,15- 9-Deoxo-9a-aza-9a-homoerythromycin A.
hexamethyl-7,16-dioxa-2-azabicyclo[11.2.1]hexadec-1-en-8-one.
c (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O- C37H70N2O12 734.96
methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy- USP Azithromycin RS
3,5,6,8,10,12,14-heptamethyl-11-[[3-amino-3,4,6-trideoxy-β-D-xylo- USP Azithromycin N-oxide RS
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-
d (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O-
methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-
Dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-
3,5,6,8,10,12,14-heptamethyl-11-[[3-formamido-3,4,6-trideoxy-β-D-xylo- hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. 3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-
e (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O- (dimethylazinoyl)-β-D-xylo-hexopyranosyl]oxy]-1-oxa-
methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12- 6-azacyclopentadecan-15-one.
hexamethyl-14-hydroxymethyl-11-[[3-dimethylamino-3,4,6-trideoxy-β-D-xylo-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. C38H72N2O13 764.98
f These impurities are controlled using the Limit of Azithromycin N-Oxide, USP N-Demethylazithromycin RS
Desosaminylazithromycin, and N-Demethylazithromycin test. They are listed here (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-
for information only. Dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-
g (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-Ethyl-3,4,10,13-tetrahydroxy-
hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-
3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-dimethylamino-β-D-xylo-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. 3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-
h (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O- methylamino-β-D-xylo-hexopyranosyl]oxy]-1-oxa-6-
methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy- azacyclopentadecan-15-one.
3,5,6,8,10,12,14-heptamethyl-11-[[3-(N-methyl)formamido-3,4,6-trideoxy-β- C37H70N2O12 734.96
D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.
i (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O- USP Desosaminylazithromycin RS
methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy- (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-Ethyl-
3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-methylamino-β-D-xylo- 3,4,10,13-tetrahydroxy-3,5,6,8,10,12,14-heptamethyl-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. 11-[[3,4,6-trideoxy-3-dimethylamino-β-D-xylo-
j (3R,4S,5S,6R,7R,9R,11S,12R,13S,14R,E)-6-[[3,4,6-Trideoxy-3-(dimeth-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-
ylamino)-β-D-xylo-hexopyranosyl]oxy]-14-ethyl-7,12,13-trihydroxy-4-[(2,6-
dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-10- one.
(hydroxyimino)-3,5,7,9,11,13-hexamethyloxacyclotetradecan-2-one. C30H58N2O9 590.79
k 9-Deoxo-9a-aza-9a-homoerythromycin A.
l (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3,3-dimethyl-α-L-
ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-

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